Stabilization of calibrators containing cytokeratin

ABSTRACT

The invention concerns calibrators or calibration solutions which are based on a human serum matrix and which are used in a method for detecting cytokeratin, a process for producing them, a method for stabilizing cytokeratin and an immunological method for detecting cytokeratin in a sample.

FIELD OF THE INVENTION

The present invention concerns calibrators or calibration solutionswhich are based on a human serum matrix and which are used in a methodfor detecting cytokeratin, a process for producing them, a method forstabilizing cytokeratin in human serum and an immunological method fordetecting cytokeratin in a sample.

BACKGROUND OF THE INVENTION

Immunological detection methods have become very important indiagnostics. They are characterized by a high specificity andsensitivity and hence they are also very suitable for detecting lowconcentrations of analytes in biological fluids. Immunological detectionmethods are of major importance particularly in the fields of infectiousdiseases, fertility and thyroid diagnostics, for metabolic diseases andfor diagnosing tumour diseases. At present carcino-embryonic antigen(CEA), alpha-fetoprotein (AFP), prostate-specific antigen (PSA) andcytokeratins (CK) are for example among the most significant tumourmarkers.

Human cytokeratins are building blocks of the intermediary filamentswhich are the major components of the cytoskeleton of epithelial cells.More than 19 cytokeratins are known of which cytokeratins 1 to 8 arereferred to as basic cytokeratins and cytokeratins 9 to 19 are referredto as acidic cytokeratins. The cytokeratins can aggregate in the cell toform tetramers. A tetramer consists of two basic and two acidiccytokeratin molecules in each case. Linear aggregation of tetramersresults in the formation of filaments. Intact cytokeratin molecules arewater-insoluble as integral components of the intermediary filaments ofepithelial cells. The complexity and composition of the cytokeratinsdiffers in the various epithelial tissues i.e. epithelial cells havecytokeratin compositions that are typical for the respective tissue. Thesoluble fragments of cytokeratin 19 (CK 19) which are also referred toas CYFRA 21-1 are particularly relevant for tumour diagnostics. Aconcentration of CYFRA 21-1 that is increased in comparison to healthypersons indicates the presence of a tumour disease. Increased valueshave previously been found in the following tumours: bronchialcarcinoma, ovarial cancer, cervix carcinoma, bladder carcinoma and intumours of the head and neck. The main indication for CYFRA 21-1 is tomonitor non-small cell bronchial carcinomas.

A method for detecting CK 19 by a sandwich ELISA technique is describedfor example in EP-A-0 460 190. In this method the body fluid sample tobe examined is incubated with at least two receptors R1 and R2, R1 andR2 being monoclonal antibodies which each detect different epitopes ofCK 19. One of the antibodies is labelled with biotin and the othercarries a different label. The sandwich complex comprising R1, CK 19 andR2 binds to a solid phase coated with streptavidin. After separating thesolid from the liquid phase, the label is measured in one of the twophases and preferably in the solid phase. The tumour marker CK 19 can bedetected in the sample on the basis of the signal that is obtained.

When carrying out such a test it is important that the measured valuethat is obtained can be at least classified qualitatively as positive(tumour marker is present) or as negative (the tumour marker is notpresent). This applies especially to the classification of measurementdata that are obtained by means of automated systems. It is often alsodesirable or necessary to quantify the concentrations of tumour marker.Hence the test system must be calibrated with reagents that contain adefined concentration of analyte before carrying out the measurements.These defined reagents are referred to in the following as calibrators.The terms calibration solution, calibration standard, standard solutionor control are used synonymously for the term calibrator.

An important requirement for a calibrator is high stability. On the onehand it is necessary to ensure the accuracy of the test, an essentially100% recovery of the analyte in the calibrator and a good signal tonoise ratio. On the other hand a reliable reproducibility of the resultof the determination must be guaranteed over a long time period. Hencecalibrators must be insensitive to their environmental conditions over atime period of several weeks i.e. to temperature, direct solar radiationon the laboratory bench, pH value, buffer conditions etc. If theconditions are unfavourable there is a risk of hydrolysis, proteolysisor denaturation of the calibrator. The use of a calibrator that is nolonger intact would lead to erroneous measurements.

Many tests are carried out in serum samples. In order to ensurecomparability of the measurements, the calibrator should therefore alsobe based on a serum matrix. The more sensitive the measuring system, thelarger the differences in measurement will become due to the jump in thematrix between sample and calibrator. It has been shown that cytokeratinis unstable in a serum matrix. When a calibrator containing cytokeratinis stored in a liquid state, the cytokeratin is destroyed and denaturedand hence such a calibrator cannot be used for a long time period.

Hence in the past the serum matrix was replaced by an artificial matrixthe composition of which mimics that of serum. The artificial matrix isgenerally based on a buffer to which various salts and proteins (forexample bovine serum albumin) are added in order to simulate as closelyas possible the natural human serum environment with regard to salt andprotein concentration as well as pH value. Although this enabled theanalyte cytokeratin to be stabilized in the calibrator, a disadvantageof this procedure is that comparability is not optimal especially withsensitive measuring systems since the underlying matrix of the samplesis human serum. There is therefore a jump in the matrix. This can leadto measuring errors with samples in the low measuring range near to thecut-off value. In extreme cases this could lead to a disparity betweenthe value determined for a positive cytokeratin signal using thecalibrator based on an artificial matrix and the value determined for ahuman serum sample even when the cytokeratin analyte is present in eachcase at a comparable concentration.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Hence the object was to provide a calibrator for methods for detectingcytokeratin which is based on a serum matrix and preferably a humanserum matrix and which is stable for a period of at least several weekseven under unfavourable ambient conditions.

The object is achieved by a calibrator whose essential components areserum, preferably human serum, cytokeratin and aprotinin. Itsurprisingly turned out that the addition of the protease inhibitoraprotinin can effectively stabilize cytokeratin. Consequently it ispossible to provide a calibrator for cytokeratin detection methods whichis manufactured on a natural serum matrix basis and in particular isbased on a human serum matrix. This avoids a jump in the matrix whenmeasuring the actual serum samples.

Furthermore it turned out that the calibrator according to the inventionhas a long shelf life and temperature stability. Thus the recovery ratefor the cytokeratin analyte dissolved in the calibrator, preferably CK19, is almost 100% in a sandwich immunoassay after stressing thelyophilised calibrator for three weeks at 30 to 40° C. Thus thecytokeratin is not destroyed and is still immunologically recognized bythe antibodies used in the test even after the time-temperature stress.The measured concentrations of cytokeratin in the various calibratorsmeasured at the start of the stability test essentially correspond tothe concentrations after the stress test. Hence the stability andprecision of the calibrator according to the invention is comparablewith the stability of a corresponding calibrator based on an artificialmatrix. Even when the calibrator is stored at 2 to 8° C. in a liquid orreconstituted form which corresponds to the usual storage temperature ofreagents in a refrigerator, a cytokeratin recovery of almost 100% wasfound even after 10 weeks. This means that the calibrator according tothe invention can still be used after long storage in a refrigeratorwithout qualitative or quantitative impairment.

The protease inhibitor aprotinin is of major importance for thesubstantially improved stability of the calibrator according to theinvention. Other protease inhibitors proved to be ineffective. Aprotininis a commercially available polypeptide which is composed of 58 aminoacids. It has an inhibitory effect on the coagulation factors XIIa, XIaand VIIIa as well as on plasmin and plasmin activators, as well astrypsin, chymotrypsin and kallikrein. surprisingly other known proteaseinhibitors have proven to be unsuitable for stabilizing the calibrator.Experiments with other substances such as detergents and salts did notresult in the desired stabilizing effect.

Aprotinin is preferably used at a concentration of at least 10 mg/l inthe calibrator. The maximum possible concentration is that whichinterferes with the test or at which turbidity starts due to the lack ofsolubility. Concentrations between 25 and 40 mg/l are particularlypreferred.

The calibrator can either be prepared in a liquid form or as alyophilisate. In order to prepare the lyophilisate, all liquid and solidcomponents are firstly mixed together or dissolved and subsequentlylyophilised. The lyophilisate is then finally used as a reconstitutedsolution i.e. it is dissolved again in liquid. Distilled water isusually used for the reconstitution i.e. to redissolve the lyophilisatesince this does not add undesired ions to the calibrator and inparticular does not change the salt concentrations. The amount of waterdepends on the desired cytokeratin concentration or on the desired fillvolume.

The calibrator according to the invention can additionally also containconventional substances known to a person skilled in the art such assalts or additional preservatives. For example N-methyliso-thiazoloneand oxypyrion are preferably added at the usual concentrations of about1 mg/l.

The calibrator according to the invention can preferably be stored in alyophilized form at 4° C. for several months without loss of quality. Itcan be stored for a period of up to 36 months at 4° C. The calibratorcan be stored in a reconstituted form for several months at 4° C.

Cytokeratin-free human serum is preferably used as the serum. This canbe obtained by affinity chromatographic processing or a serum is usedwhich is free of cytokeratin from the beginning which has to bedetermined by appropriate screening methods.

The invention additionally concerns a process for producing thecalibrator according to the invention. The calibrator is preferablyproduced by the following steps

a) mixing serum with aprotinin

b) filtering the solution

c) dissolving the cytokeratin in water

d) mixing the solution from step b) with the dissolved cytokeratin fromstep c)

e) lyophilizing the solutions from d)

f) dissolving the lyophilisate in water before use.

If necessary the pH value can be adjusted in step d) to pH 7 to 8,preferably 7.2.

A further subject matter of the invention is also a process forproducing stabilized cytokeratin which is characterized in that theprotease inhibitor aprotinin is added to the preferably purifiedcytokeratin. The stabilized cytokeratin produced in this manner ispreferably used in a calibrator.

Another subject matter of the invention is the use of aprotinin tostabilize cytokeratin and in particular CK 19.

A subject matter of the invention is also a method for stabilizingcytokeratin in serum, preferably human serum which is characterized inthat aprotinin, preferably at a concentration of at least 10 mg/l, isadded to the serum.

A further subject matter of the invention is an immunological method fordetermining cytokeratin in a sample and in particular to determine CK19. The method is characterized in that the signal determined for thesample is compared with the signal which is obtained with the aid of thecalibrator according to the invention, the calibrator being measuredusing the same method as for the sample.

Such a method for determining cytokeratin and preferably for determiningCK 19 and the calibrator is preferably carried out using the followingsteps:

a) reacting the sample with a first binding partner that is specific forcytokeratin and carries a group capable of binding to a solid phasewhich can be used to bind it to a solid phase,

b) reacting this solution with a further binding partner which carries alabel

c) binding the immune complex that is formed to a solid phase, wherebythe solid phase can already be present in step a)

d) separating the solid from the liquid phase

e) detecting the label in one of the two phases.

f) comparing the measured values for the calibrator with the value forthe sample and quantification.

The method can also be carried out as a competitive test by methodsknown to a person skilled in the art.

The binding partners are preferably monoclonal or polyclonal antibodiesor fragments thereof such as F(ab′)₂, Fab′ or Fab fragments which canspecifically immunologically recognize and bind cytokeratin and inparticular CK 19. The antibodies are produced by methods familiar to aperson skilled in the art. Antibodies are also included which have beenproduced by modifying the antibodies for example by genetic engineering.The term antibody includes all aforementioned meanings for bindingpartners.

The first specific binding partner for cytokeratin can either bedirectly bound to the solid phase or the binding to the solid phaseoccurs indirectly via a specific binding system. The direct binding ofthis binding partner to the solid phase occurs according to methodsknown to a person skilled in the art. If the binding is indirect via aspecific binding system, then the first binding partner is a conjugatecomprising an antibody to cytokeratin and one reaction partner of aspecific binding system. In this case a specific binding system isunderstood as two partners that can specifically react one another. Thebinding capability can be based on an immunological reaction or onanother specific reaction. A combination of biotin and avidin or biotinand streptavidin is preferably used as the specific binding system.Other preferred combinations are biotin and antibiotin, hapten andanti-hapten, Fc fragment of an antibody and antibody to this Fc fragmentor carbohydrate and lectin. One of the reaction partners of the specificbinding pair is then a part of the conjugate.

The other reaction partner of the specific binding system for the firstbinding partner is present as a coating on the solid phase. The otherreaction partner of the specific binding system can be bound to aninsoluble carrier material by conventional methods known to a personskilled in the art. In this case a covalent as well as an adsorptivebinding is suitable.

Suitable solid phases are test tubes or microtitre plates made ofpolystyrene or similar plastics whose inner surface is coated with areaction partner of the specific binding system. Other suitable andparticularly preferred solid phases are particulate substances such aslatex particles, magnetic particles, molecular sieve materials, glassbeads, plastic tubes etc. Porous layer-like carriers such as paper canalso be used as carriers. Magnetic particles, so-called beads, areparticularly preferably used and are in turn coated with the appropriatebinding partner of the specific binding system described above. In orderto carry out the detection reaction, these microparticles can then beseparated from the liquid phase after completion of the test reactionfor example by filtration, centrifugation or by a magnet in the case ofmagnetic particles.

The specific binding reactions between the antibodies to cytokeratin andcytokeratin can be detected in various ways. Usually one binding partnerof the specific binding reaction is labelled. Common labels arechromogens, fluorophores, substances capable of chemiluminescence orelectrochemiluminescence, radioisotopes, haptens, enzyme labels orsubstances which can in turn form a specific binding pair such asbiotin/streptavidin.

All biological fluids familiar to a person skilled in the art can beused as samples to carry out the method for the detection ofcytokeratin. Body fluids are preferably used as the sample such as wholeblood, blood serum, blood plasma, urine or saliva, particularlypreferably blood serum.

The invention is further elucidated by the following examples.

EXAMPLE 1 Production of the Calibrator According to the Invention

The stated identification numbers (Id No.) refer to catalogue numbersfrom Boehringer Mannheim GmbH, Germany. In order to prepare 1 litre CKcalibrator (CK concentration 40 ng/ml) the following are mixed together.

A: commercial human serum made cytokeratin-free by affinitychromatography

B: 34 mg aprotinin, Id.No: 0236632-001

C: 1 g N-methylisothiazolone HCL, Id.No: 1085901-105

D: 1 g oxypyrion, Id.No: 1085913-05

E: 7.31 g cytokeratin from a stock solution having a concentration of0.05 μg/ml containing 941 g A+B+C+D

The human cytokeratin Cyfra 21-1 that was used corresponds to thecytokeratin from the human cell line MCF-7 of the Cyfra 21-1 CalSet,calibration set from the Elecsys® CYFRA 21-1 immunoassay from BoehringerMannheim GmbH, Germany Id.No: 1820974.

EXAMPLE 2 Stressing the Calibrator at 35° C.

The procedure is according to the Elecsys® Cyfra 21-1 method fromBoehringer Mannheim GmbH (Id.No. 1 820 966). The Elecsys® test procedureis based on the biotin/streptavidin technology. The test principle is a1-step sandwich ELISA using two antibodies that recognize differentepitopes on the analyte (cytokeratin). The solid phase comprisesmagnetic latex beads coated with streptavidin to which a biotinylatedantibody that is specific for cytokeratin binds. The bound tumour markeris detected after separation of the solid from the liquid phase bymeasuring the electrochemiluminescence which is generated by a secondspecific antibody for cytokeratin that is labelled with a rutheniumcomplex.

The following raw materials are used:

R1 (first antibody, biotinylated Fab fragment)

MAB<CK19>M-KS19.1-Fab(DE)-Bi(DDS); concentration 1.5 μg/ml

R2 (second antibody, ruthenylated IgG):

MAB<CK19>M-BM19.21-IgG-BPRU); concentration 2.1 μg/ml

90 μl R1 and 90 μl R2 are incubated together with streptavidin-coatedmagnetic beads according to the instructions of the manufacturer of theElecsys®2010 instrument from Boehringer Mannheim GmbH, Germany andmeasured.

The freshly dissolved calibrator according to the invention containingcytokeratin was compared to a calibrator according to the invention thatwas stressed for 3 weeks in a lyophilised state at 35° C. andsubsequently reconstituted. The results are shown in table 1.

TABLE 1 (example 2): Human matrix master calibrators, freshly dissolvedand after a three week stress at 35° C. after a 3 week stress at 35° C.freshly dissolved conc. recovery conc. recovery (ng/ (%) comp. MC(ng/ml) (%) ml) to fresh 1 0.0 / 0.0 / 2 0.88 100 0.87 99 3 7.57 1007.10 94 4 18.13 100 17.19 95 5 60.85 100 57.62 95 6 125 100 119 95 7 302100 296 98 8 450 100 430 96 9 613 100 604 96 MC: master calibrator:calibrator that is used to calibrate a new reagent lot before dispatchrec: recovery

The table shows that the stressed calibrator also has a good and almost100% recovery. The deviation is in each case less than 10% (largestdeviation 6%). Hence the calibrator according to the invention is stabletowards a high temperature over a long time period and yields reliablecalibration values even after stress. Even if the calibrator isaccidentally stored incorrectly it can be used further withoutreservation and without impairment of quality.

EXAMPLE 3 Long-term Stress of the Calibrator at 4° C. (Liquid)

A comparison was made between freshly dissolved calibrator andcalibrator which was stored before the measurement in a liquidreconstituted form at 4° C for 10 weeks. The measurements are carriedout as described in example 2. The results are shown in table 2.

TABLE 2 (example 3): Human matrix standard, freshly dissolved and aftera 10 week storage at 4° C. after 10 week storage at 4° C. freshlydissolved conc. recovery Stan- conc. recovery (ng/ (%) comp. dard(ng/ml) (%) ml) to fresh A 0.20 / 0.44 / B 3.33 100 3.13 94 C 8.00 1007.85 98 D 18.84 100 17.81 95 E 54.52 100 51.68 95 F 100.89 100 95.53 95

The calibrator according to the invention also exhibits good results ina liquid form with regard to signal recovery and concentration recoveryafter a stress for 10 weeks at 4° C.

EXAMPLE 4 Long-term Stability of the Calibrator: 24 Months at 4° C./24Months −20° C.

A comparison was made between the calibrator which was stored before themeasurement for 24 months at 4° C. and the calibrator which was storedfor 24 months at −20° C. (each as a lyophilisate). The procedure is asdescribed in example 2. The results are shown in table 3.

TABLE 3 (example 4) Human matrix calibrators after 24 months storage at−20° C. and +4° C. 24 months at 24 months at +4° C. −20° C. conc.recovery (%) stan- conc. recovery (ng/ml) relative dard (ng/ml (%) ml)to fresh MC1 0.0 / 0.0 / MC2 3.71 100 3.85 104 MC3 9.01 100 9.04 100 MC440.11 100 39.82  99 MC5 394.8 100 395.0 100

There is a good recovery in both stress methods. Calibrator containingcytokeratin can be stored without reservation in a lyophilized form overtime periods of several years at +4° C. or −20° C. without significantimpairment to the quality.

EXAMPLE 5 Comparison of Measured Values of Tumour Marker Controls:Calibration of the Measuring Instrument on an Artificial Matrix andHuman Serum Basis

The Elecsys® measuring instrument is calibrated before carrying out themeasurements with the calibrator containing cytokeratin based on anartificial matrix and subsequently the tumour marker controls aremeasured on the basis of these calibration values. The values found forthis measurement are set at 100% recovery according to definition.Subsequently the same instrument is calibrated with the human serumbased calibrator according to the invention and again the tumour markercontrols are measured on the basis of these calibration values. Theresults are shown in table 4.

The tumour marker controls TMC I or II and PCT I or II are based onprocessed human serum and contain the cytokeratin Cyfra 21-1 with targetvalues for two concentration ranges (I and II) in addition to othertumour markers such as PSA and AFP.

TABLE 4 (example 5) Comparison between tumour marker controls;cytokeratin calibration based on an artificial matrix compared to ahuman serum based calibration calibration with calibration withcalibrator based on calibrator based an artificial matrix on human serumtumour conc. conc. marker (ng/ recovery (ng/ recovery control ml) (%)ml) (%) TMCI 4.63 100 5.07 110 TMCII 28.5 100 29.8 105 PCTI 4.99 1005.23 105 PCTII 30.7 100 31.7 103

It can be seen that the deviations between measurements of tumour markercontrols based on calibrations using the calibrators based on anartificial or human matrix are no more than 10%. The values obtained forthe tumour marker controls are thus independent of the calibrator usedto calibrate the instrument. The calibrator based on human serum canthus replace the calibrator based on an artificial matrix.

What is claimed is:
 1. A calibrator for use in the detection ofcytokeratins, the calibrator comprising: a serum, a known concentrationof cytokeratin, and aprotinin.
 2. The calibrator of claim 1, whereinsaid serum comprises a natural serum.
 3. The calibrator of claim 2,wherein said natural serum comprises human serum.
 4. The calibrator ofclaim 3, wherein said human serum is substantially cytokeratin-free. 5.The calibrator of claim 1, wherein said aprotinin is present in aconcentration of at least 10 mg/l.
 6. The calibrator of claim 1, whereinsaid aprotinin is present in a concentration from about 25 mg/l to about40 mg/l.
 7. The calibrator of claim 1, wherein said cytokeratincomprises CK
 19. 8. The calibrator of claim 1, further comprising apreservative.
 9. The calibrator of claim 8, wherein said preservativecomprises n-methylisothiazolone.
 10. The calibrator of claim 8, whereinsaid preservative comprises oxypyrion.
 11. The calibrator of claim 1,wherein the calibrator is in a liquid form.
 12. The calibrator of claim1, wherein the calibrator is a lyophilisate.
 13. The calibrator of claim1, wherein the calibrator is stable for three weeks at 30° C. to 40° C.14. The calibrator of claim 1, wherein the calibrator is stable for atleast ten weeks at 2° C. to 8° C.
 15. The calibrator of claim 1, whereinthe calibrator is stable for up to 36 months at 4° C.
 16. A method forstabilizing a cytokeratin calibrator wherein said calibrator contains aserum and a known concentration of cytokeratin, said method comprisingadding aprotinin to the calibrator.
 17. The method of claim 16, whereinthe resulting calibrator has an aprotinin concentration of least 10mg/l.
 18. The calibrator of claim 16, wherein the resulting calibratorhas an aprotinin concentration from about 25 mg/l to about 40 mg/l. 19.A process for producing a cytokeratin calibrator, the processcomprising: mixing a serum with aprotinin to form a solution; dissolvinga known concentration of cytokeratin in water; mixing said solution andsaid dissolved cytokeratin to form the cytokeratin calibrator.
 20. Theprocess of claim 19, further comprising lyophilizing said calibrator.21. A method for determining the concentration of cytokeratin in asample, the method comprising: measuring a first value relating to theconcentration of cytokeratin in the sample; measuring a second valuerelating to the concentration of cytokeratin in a calibrator using thesame methodology used to measure the first value, the calibratorcomprising a serum, a known concentration of cytokeratin and aprotinin;and comparing the measured first value to the measured second value todetermine the concentration of cytokeratin in the sample.
 22. The methodof claim 21, wherein the cytokeratin in said sample and said calibratorcomprises CK
 19. 23. The method of claim 21, wherein said calibratorcomprises at least 10 mg/l of aprotinin.
 24. The calibrator of claim 21,wherein said calibrator comprises about 25 mg/l to about 40 mg/l ofaprotinin.